The goal of OSI Pharmaceuticals Inc. has been to discover small molecular weight compounds that promote increased fetal gamma-globin gene expression in adult red blood cells for the treatment of sickle cell disease and beta-thalassemia. In man, there is compelling evidence, genetic and pharmaceutical, that increasing expression of the gamma-globin gene has a dramatic clinical benefit on both diseases. Pharmacological agents which inhibit protein deacetylation, inhibit DNA methylation or induce specific erythroid cell stress pathways have been shown to increase HbF levels in vitro and in vivo. Our investigations indicate that other mechanisms of increasing gamma-globin gene expression are likely. High-throughput robotic screening of approximately 140,000 chemical compounds and natural products reveal at least 9 distinct classes of relatively non-cytotoxic chemical compounds which induce HbF. Numerous analogues also have been evaluated. One of these compounds, OSI-2040, is a novel histone deacetylase inhibitor and transcription factors which mediate its activity recently were identified. The mechanisms by which the remaining distinct compound classes act to affect HbF induction yet are undetermined. Here we propose a series of experiments designed to identify molecular targets of drug action. Several focused and general approaches to examining mechanisms of action of our HbF inducing compounds are proposed,using both well established methods and relatively advanced affinity chromatography and mass spectrometry techniques to characterize compound activity and mechanisms of action. Information regarding mechanism of action are important to the advancement of most optimal and diverse compounds through animal testing, toxicology and to clinical evaluation. PROPOSED COMMERCIAL APPLICATIONS: The medical need for effective, non-toxic therapeutics for sickle cell disease of beta-Thalassemia is largely unmet. In the U.S. alone, there are approximately 80,000 patients with sickle cell disease, and an currently available treatments are both inadequate and expensive. The cost of treating a sickle cell patient in the U.S. has been estimated to be approximately S60,000 per year and thalassemia to be approximately $ 100,900 per year. The number sickle cell, beta and alpha thalassemia patients in emerging economies world wide is large.